High density lipoprotein (HDL) and its major protein constituent, apolipoprotein (apo)A-I, may play critical roles in the prevention of cholesterol accumulation in blood vessels that can lead to human cardiovascular disease, which claims nearly a million lives per year in the United States. Unfortunately, relatively little is known about the molecular basis for the cardio-protective effects of HDL. A prominent obstacle in the way of a detailed understanding of these effects is the lack of information on the structure of apoA-I in HDL. We propose to test the hypothesis that the structure of apoA-I in spherical human plasma HDL particles is related to that in the simplest discoidal particles that can be created in vitro. The approach will be to take advantage of the geometric constraints inherent to the edge of reconstituted discoidal MDL particles to generate a highly detailed model of apoA-I organization on these particles. Using this structure as a benchmark, changes in spatial relationships between regions of apoA-I will then be monitored as the complexity of the particles is systematically increased from discs, to well-defined reconstituted spherical particles, and finally to isolated human HDL particles. Two complementary approaches will be used to monitor the distance parameters within and between apoA-I molecules on the particles. These are: A) the use of a comprehensive battery of tryptophan and cysteine mutants of apoA-I to study fluorescence energy transfer, and B) the application of a novel mass spectrometry/peptide mapping technique that takes advantage of reversible thiol cross-linkers to determine the proximity of various regions of apoA-I in lipoproteins. These methods will be used to generate a detailed "proximity map" of HDL-bound apoA-I in different particle morphologies. This information will provide a basis for highly targeted mutagenesis strategies resulting in apoA-I variants that can be used in vivo to dissect out the cardio-protective functions of apoA-I.